The ADP-ribosylation factors (ARFs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of ARF6, an ARF that affects plasma membrane (PM) traffic and the actin cytoskeleton. ARF6 regulates the movement of PM into and out of a novel, endosomal recycling pathway. The return of membrane to the PM requires ARF6 activation, occurs at discrete sites along the peripheral edges of cells and is associated with the formation of actin containing protrusions and membrane ruffling. Formation of protrusions is exaggerated in HeLa cells overexpressing ARF6 upon the addition of aluminum fluoride (AlF), an activator of heterotrimeric G proteins. The AlF treatment results in an accumulation of ARF6 at the PM, formation of protrusive structures, and a block in internalization of PM into the endosomal compartment. These are also characteristics of the effect of expressing the constitutively active mutant of ARF6 in cells. To investigate whether a G protein was responsible for this effect of AlF and thus a potential upstream regulator of ARF6, we cotransfected HeLa cells with ARF6 and constitutively active, candidate G alpha proteins. G alpha q, but not Gs, Gi2, or G12, appears to specifically activate ARF6 and recreates effects observed with AlF. We are currently investigating the mechanism whereby Gq maintains ARF6 in the activated state. The ADP-ribosylation factors (ARFs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of ARF6, an ARF that affects plasma membrane (PM) traffic and the actin cytoskeleton. ARF6 regulates the movement of PM into and out of a novel, endosomal recycling pathway. The return of membrane to the PM requires ARF6 activation, occurs at discrete sites along the peripheral edges of cells and is associated with the formation of actin containing protrusions and membrane ruffling. Formation of protrusions is exaggerated in HeLa cells overexpressing ARF6 upon the addition of aluminum fluoride (AlF), an activator of heterotrimeric G proteins. We have found that the target of AlF in our cells is G alpha q since the acute stimulation of protrusion formation induced by AlF can be mimicked in cells cotransfected with ARF6 and the constitutively active mutant of Gq, Q209L. The AlF- and Gq induced protrusions are sensitive to inhibitors of PLA2, but insensitive to inhibitors of PI3kinase, protein kinase C, and calcium. As Gq has been implicated as an activator of PLC, our inhibitor studies suggest that Gq affects ARF6 independently of PLC. Besides inducing protrusions, Gq (Q209L) expression alone influences the ARF6 regulated membrane trafficking pathway, resulting in accumulation at the PM of ARF6 and other membrane proteins that normally cycle through the ARF6 pathway. In order to identify domains in ARF6 responsible for its cellular localization and function, we have constructed a chimera between ARF1 and ARF6 and expressed it in HeLa cells. We have found the carboxyl-terminal half of ARF6 contains information sufficient to target the chimera to the PM and endosome, and not to the Golgi complex (where ARF1 localizes) but this chimera does not form protrusions in response to AlF, an ARF6 effector function. We have identified 2 amino acid residues (QS) in the amino-terminal half of ARF6 that when substituted into the 1-6 chimera results in a gain-of-function, where protrusions now form. Mutation of the QS in wild type ARF6 results in a loss of function, where the mutant ARF6 cannot form protrusions. The QS residues are positioned near the switch 1 region of ARF6 and are conserved among nearly all ARF6 homologues whereas EI is found in all other ARF proteins at this position. Mapping this effector domain to these two amino acids in ARF6 will facilitate the identification of interacting effector molecules and development of inhibitory peptides or antibodies that can be used to block ARF6 function. We have begun to characterize potential interacting proteins that we have identified through a yeast two-hybrid screen using the consitutively active ARF6 mutant, Q67L, as bait. - ADP-ribosylation factor, ARF6, ARF1, GTPase, membrane traffic, actin cytoskeleton